A great deal of research has been directed to the development of accurate techniques for determining the presence of organic materials such as drugs, contaminants, pollutants, and the like in substances of interest such as food, soil, and bodily fluids. For example, pathological or other conditions in human beings and animals are often detected by performing immunoassays on samples of bodily fluids such as urine or blood serum. Immunoassays are based on the capacity of a first compound, known as a ligand, to recognize or bind a second compound, known as a receptor, having a specific spatial and/or polar organization. Typically, immunoassays are used for the detection of antibodies or antigens in bodily fluids.
Antigens are foreign substances which, when introduced into a higher animal, bring about the formation of antibodies which react with the antigens to initiate protection against infection or disease. The presence of an antigen or an antibody can be confirmed or determined by adding the corresponding antibody or the corresponding antigen to a sample of bodily fluid. The presence or absence of the antibody or antigen in the sample is usually established by detecting the occurrence or nonoccurrence of a reaction between the specific ligand/receptor pair, which usually manifests itself by insolubility or agglutination.
Because most ligand/receptor pairs are detected only with difficulty, it is frequently necessary to use certain inert carrier moieties to facilitate their detection. For example, in most latex agglutination techniques the receptor is covalently bound to discrete latex particles having diameters on the order of about 0.01 to about 100 micrometers. These particles are cross-linked or otherwise aggregated by the complementary ligand. The agglutination of such particles into relatively large aggregates or clumps is then observed.
The different types of latex agglutination techniques presently known in the art may be categorized into three basic classes based upon the particular method employed for detecting ligand/particle aggregates. The techniques of the first class involve centrifugation. For example, U.S. Pat. No. 4,738,932 in the name of Yabusaki discloses a centrifugation technique which involves rotating an agglutination slide on a serological rotator and then using a magnifier to examine slide wells for agglutination.
The second class comprises techniques which detect aggregates by particle counting. For example, Masson, et al., Methods in Enzymology, 1981, 74, 106-139, disclose an immunoassay technique in which a complicated device which uses forward light scattering is employed to count unaggregated particles. Thus, both the centrifugation and particle counting techniques have the disadvantages of complicated, time-consuming procedures and expensive, highly specialized devices.
The techniques of the third class are those in which agglutination is detected visually. However, since the average human eye can only detect particles down to about 40 micrometers in diameter, most visual agglutination tests must produce relatively large aggregates which typically require an undesirably long time to form. U.S. Pat. No. 4,459,361 in the name of Gefter discloses a somewhat improved type of visual technique which involves visual detection of unaggregated particles rather than aggregated particles. In the technique according to Gefter, both the ligand and the receptor are separately immobilized on latex particles and then mixed with a sample of bodily fluid suspected to contain the ligand. According to Gefter, the ligand in the sample competes with the ligand-bearing particles for the receptor sites on the receptor-bearing particles. To the extent that the ligand contained in the sample binds to the receptor-bearing particles, the ligand-bearing particles fail to aggregate. Thus, when such a mixture is exposed to a filter having a controlled pore size there is a substantial increase in the amount of unaggregated, ligand-bearing particles which pass through the filter. It is the presence of these unaggregated particles which is then detected.
According to Gefter, the amount of unaggregated particles which pass through the filter is proportional to the amount of ligand in the sample. Further, Gefter states that the number of unaggregated particles is sufficient to be visible to the naked eye, and that this visibility can be enhanced by the selection of the size, color, optical density, or fluorescence of the particles.
However, the technique disclosed by Gefter has a number of serious shortcomings. For example, the technique requires that both ligand- and receptor-bearing particles be prepared, thus adding considerable time and expense. While Gefter asserts that the unaggregated, ligand-bearing particles can be detected with the naked eye, the provided examples do not detect such particles visually, but rather with sophisticated spectrophotometric devices.
Accordingly there exists a long-felt need for relatively simple, inexpensive techniques for the accurate detection of biologically important chemical compounds present in bodily fluids and in other sample fluids.